JP2022074265A - Reagent for immunonephelometry and kit therefor - Google Patents

Abstract

To provide a reagent for immunonephelometry that can accurately measure an object to be measured in a sample and a kit therefor.SOLUTION: The present invention discloses a reagent for immunonephelometry that comprises a carrier particle that is sensitized by a specific binding substance to an object for measuring, and casein or a salt thereof. There is also provided a kit therefor.SELECTED DRAWING: None

Description

The present invention relates to a reagent for measuring turbidimetry and a measurement kit of a measurement object in a test sample.

As an immunological measurement method using a specific binding reaction between an antigen and an antibody, many measurement methods such as an enzyme immunoassay method, a fluorescent enzyme immunoassay method, an immunoturbidimetric method, an immunopistosis fistula method, and an immunochromatography method are known. ing.

In particular, the immunoturbidimetry method is widely used because it is easy to operate and the measurement can be automated. The immunoturbidimetric method is a method of detecting an object to be measured by reacting carrier particles sensitized with an antibody specific to the object to be measured and measuring the absorbance of the complex aggregate generated by the specific binding reaction. Is.

However, the immunological measurement method has a problem that the measurement target cannot be accurately measured due to a non-specific reaction. In order to prevent non-specific reactions, immunoassay reagents containing various proteins, surfactants, salts and the like have been proposed. For example, a method using 900,000 daltons or more of casein to prevent a non-specific reaction of a labeled ligand (Patent Document 1), a non-specific reaction inhibitor consisting of a protein, a polymer, a salt, or a combination thereof is used. A method (Patent Document 2) and the like can be mentioned.

Japanese Unexamined Patent Publication No. 2004-117341 Japanese Unexamined Patent Publication No. 2010-127827

An object of the present invention is to provide a reagent for measuring turbidimetry and a measurement kit capable of accurately measuring an object to be measured in a test sample.

As a result of diligent studies to solve the above problems, the present inventors have sensitized a carrier particle to a specific binding substance to the measurement target, and the immunological ratio of the measurement target containing casein or a salt thereof. The present invention has been completed by finding that the above-mentioned problems can be achieved by the turbidity method measuring reagent and the measuring kit.

The present invention comprises the following.
1. 1. A reagent for measuring an immunoturbidimetric method of a measurement target, which comprises carrier particles sensitized to a specific binding substance to the measurement target and casein or a salt thereof.
2. 2. The reagent for measuring turbidimetry according to item 1 above, wherein the specific binding substance is an antibody or antigen that specifically binds to an object to be measured.
3. 3. The reagent for measuring an immunoturbidimetric method according to the above item 1 or 2, wherein the carrier particles are any one of cellulose particles, latex particles, metal nanoparticles, and gelatin particles.
4. 6. Reagent for measuring immunoturbidimetric method.
5. The reagent for measuring turbidimetry according to any one of the above items 1 to 4, further comprising a saccharide and / or a surfactant.
6. The reagent for measuring turbidimetry according to any one of the above items 1 to 5, wherein the measurement target is a C-reactive protein.
7. The reagent for measuring turbidimetry according to any one of the above items 1 to 6 is applied to a measuring device, and the measuring reagent is kept in a dry state until it is used for measurement. , A kit for measuring the object to be measured by the immunoturbidimetry method.
8. A reagent for measuring immunoturbidimetric method of the object to be measured, which contains carrier particles sensitized to a specific binding substance to the object to be measured and casein or a salt thereof, is applied to the device for measurement until it is used for measurement. A measurement kit by an immunoturbidimetric method of an object to be measured, characterized in that the measurement reagent is kept in a dry state.
9. The measurement reagent applied to the measurement kit according to the above item 7 or 8 is mixed with the test sample, and is generated by a specific binding reaction between the carrier particles contained in the measurement reagent and the measurement target in the test sample. A method for measuring an object to be measured by measuring the composite aggregate.
10. A method for manufacturing a kit for measuring an object to be measured by the immunoturbidimetry method, which comprises the following steps:
1) A step of applying a reagent for measuring an immunoturbidimetric method of a measurement target, which contains carrier particles sensitized to a specific binding substance to the measurement target and casein or a salt thereof, to the measurement device;
2) A step of drying the measurement reagent applied to the measurement device.

According to the immunoturbidimetric measurement reagent and measurement kit of the measurement target, which contains the carrier particles sensitized to the specific binding substance to the measurement target of the present invention and casein or a salt thereof, the carrier particles are not present. The peculiar reaction can be efficiently suppressed, and the measurement target in the test sample can be accurately measured.

It is a figure which shows the relationship between the mean value (measured value) converted into the concentration, CRP-free serum, and the CRP-controlled sample of CRP known concentration (0.4, 0.8, 1.6, 3.0mg / dL) in a graph (Example 2).

Hereinafter, the present invention will be described in detail.

(1) Reagent for measurement The "reagent for measurement" of the present invention is characterized by containing carrier particles sensitized to a specific binding substance to an object to be measured, and casein or a salt thereof.

As used herein, the "carrier particles" are carrier particles that can be used for measurement by the turbidimetry method, and are not particularly limited as long as they can sensitize a specific binding substance to the object to be measured. Examples of the carrier particles include cellulose particles, latex particles, gold nanoparticles, gelatin particles, liposomes, microcapsules, particles such as erythrocytes, and the like, preferably cellulose particles, latex particles, gold nanoparticles, gelatin particles, and the like. Cellulous particles are preferred.

In the measurement by the turbidimetry method, the average particle size of the carrier particles is not particularly limited. Ease of forming composite aggregates produced by the specific binding reaction between the carrier particles sensitized to the specific binding substance to the measurement target and the measurement target in the test sample, and the measurement of the produced complex aggregates. The average particle size of the carrier particles is preferably 50 nm to 1000 nm, and particularly preferably 100 nm to 400 nm for reasons such as ease of use. As the carrier particles, two or more types of carrier particles having different particle sizes, shapes, and the like may be used.

As used herein, the “specific binding substance” is not particularly limited as long as it is a substance that causes a specific binding reaction with respect to the object to be measured. A substance that can be used for an antigen-antibody reaction is preferable, and an antibody or an antigen that specifically binds to an object to be measured is particularly preferable. When the object to be measured is an antigen, an antibody corresponding to the antigen can be mentioned, for example, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a human antibody or a single-stranded antibody (scFv), and a fragment (Fab) of these antibodies. , F (ab') 2), Fab', Fv, sFv, dsFv, etc.) can be appropriately selected and used. In addition, a derivative can be used by binding a protein or a small molecule compound to an antibody or an antibody fragment. The origin of the antibody is not particularly limited, and examples thereof include mammals (mouse, rabbit, rat, sheep, goat, horse, etc.), birds (chicken, quail, pheasant, ostrich, duck, etc.) and the like. When the object to be measured is an antibody, it may be an antigen against the antibody, and for example, the antigen against the above-exemplified antibody may be used as the antigen.

The "casein or a salt thereof" in the present invention is used as a blocking agent for suppressing a non-specific reaction of carrier particles. For example, αs-casein, β-casein, κ-casein and the like can be mentioned, and sodium hydroxide may be added to these caseins. The casein or a salt thereof is preferably casein Na.

Additives such as saccharides and surfactants may be further added to the measurement reagent of the present invention. Examples of the saccharide include sucrose, sorbitol, mannitol, trehalose, lactose, xylitol, galactose, allose, glucose, altrose and the like, and sucrose, sorbitol and mannitol are particularly preferable. Examples of the surfactant include amphoteric surfactants and nonionic surfactants, and examples thereof include CHAPS, CHAPSO, ASB-14, C7Bz0, Tween20, Briji35, I GEPAL CA-630, and digitonin. CHAPS and Briji35 are preferred.

(2) Method for Preparing Reagent for Measurement In the reagent for measurement of the present invention, the sensitization to the carrier particles of the specific binding substance to the object to be measured is not particularly limited, but can be prepared, for example, as follows. The carrier particles can be prepared by adding a specific binding substance such as an antibody to the carrier particles, stirring the mixture in an aqueous solvent, and then washing the carrier particles sensitized with the specific binding substance to the object to be measured by centrifugation.

The reagent for measurement of the present invention can be prepared by adding various aqueous solvents to the carrier particles sensitized by the binding substance and adding casein or a salt thereof. Further, the above additives and specific binding substances may be added. Examples of the aqueous solvent include purified water, physiological saline, Tris buffer, phosphate buffer and the like, and the pH of the aqueous solvent is generally pH 5 to 10, for example. The specific binding substance not only sensitizes the carrier particles, but also adjusts the sensitivity of the carrier particles sensitized to the specific binding substance to the measurement target and the specific binding reaction of the measurement target in the test sample. It can be added to the measuring reagent.

The liquid reagent for measurement of the present invention prepared by the above method contains 0.10 to 0.50 w / v%, preferably 0.15 to 0.25 w / v% of carrier particles sensitized to a specific binding substance to the object to be measured. It is contained and contains 0.05-5.00w / v%, preferably 0.10-1.00w / v% of casein or a salt thereof.

(3) Measurement kit The present invention also includes a "measurement kit" by the immunoturbidimetric method of the object to be measured. The kit is characterized in that the measuring reagent of the present invention is applied to a measuring device and dried, and the measuring reagent is kept in a dry state until it is used for measurement.

In the measurement kit of the present invention, the "measurement device" is the measurement of the present invention containing at least a sample supply unit for supplying a test sample and carrier particles sensitized to a specific binding substance to the object to be measured. Includes the reaction part to which the reagent is applied. Further, it is preferable to include a route for introducing the test sample supplied to the sample supply unit to the reaction unit as a component. The reaction portion preferably has an upper portion and a lower portion as a portion to which the measurement reagent of the present invention is applied, and may have a shape that allows the measurement reagent and the test sample to be mixed, and is preferably concave, for example. ..

(4) Method for manufacturing a measurement kit The present invention also includes a "method for manufacturing a measurement kit" for a measurement object by the turbidimetry method.
The method for producing a measurement kit of the present invention is characterized by comprising a step of applying the measurement reagent of the present invention to a measurement device and a step of drying the measurement reagent of the present invention.

In the "method for manufacturing a measurement kit" of the present invention, it is preferable to apply the measurement reagent of the present invention to the reaction part of the measurement device, and after the measurement reagent of the present invention is applied to the measurement device in a liquid state. It may be allowed to be in a dry state, or the measurement reagent of the present invention in a dry state may be applied to the measurement device. Examples of the method for drying the measurement reagent of the present invention include natural drying and freeze-drying.

(5) Measurement Method The present invention also includes a "measurement method for an object to be measured". In the measuring method of the present invention, a measuring reagent applied to the measuring kit of the present invention and a test sample are mixed, and carrier particles contained in the measuring reagent and sensitized to a specific binding substance to a measurement object are sensitized. This is a method of measuring an object to be measured by measuring a complex aggregate generated by a specific binding reaction of the object to be measured in the test sample.

Hereinafter, the measuring method of the present invention will be specifically described.
1) When the test sample is supplied to the sample supply unit of the measurement device included in the measurement kit of the present invention, the test sample moves to the reaction unit, for example, through the path of the measurement device. Since the measurement reagent of the present invention is applied to the reaction section, the measurement reagent of the present invention and the test sample are mixed by dissolving the measurement reagent of the present invention from a dry state. The reagent for measurement of the present invention may be applied only to the upper part of the reaction part, only to the lower part, or may be applied to both.
2) By mixing the measurement reagent of the present invention and the test sample, a composite aggregate is generated by a specific binding reaction between the carrier particles contained in the measurement reagent of the present invention and the measurement target in the test sample. ..
3) By irradiating the solution containing the generated complex aggregate with light and measuring the absorbance, the object to be measured contained in the test sample can be detected.

In the measuring method of the present invention, the temperature at which the measuring reagent of the present invention and the test sample are reacted in the reaction section is, for example, 0 to 50 ° C, preferably 4 to 40 ° C. When irradiating the solution containing the generated composite aggregate with light, it is preferable to irradiate the reaction portion with light, and to irradiate the light, for example, a spectrophotometer or the like can be used.

The "test sample" to be used for the measurement method of the present invention may be a sample itself obtained from a living body or may be prepared from a sample. For example, the test sample includes a blood sample, a body fluid, a urine sample and the like. Examples of the blood sample include whole blood, serum, plasma and the like.

In the present specification, the "measurement object" includes an object to be inspected in a general clinical examination or the like, and specifically, various components contained in blood, urine, saliva, secretions, etc., tissues, feces, etc. Examples thereof include various components contained in an extract or the like from a solid substance such as. Specific examples of objects to be measured include CRP, albumin, AST, ALT, γGTP, hemoglobin, hemoglobin A1c, myoglobin, transferase, lactoferrin, cystatin C, ferritin, α-fetoprotein, cancer fetal antigen, CA19-9, Proteins such as prostate-specific antigens, fibrinolytic products (FDPs), pepsinogens I and II, collagen; lipid proteins such as high specific gravity lipoproteins, low specific gravity lipoproteins, and ultralow specific gravity lipoproteins; nucleic acids such as deoxyribonucleic acid and ribonucleic acid. Enzymes such as alkaline phosphatase, lactic acid dehydrogenase, lipase, amylase; immunoglobulins such as IgG, IgM, IgA, IgD, IgE; hepatitis B virus, hepatitis C virus, human immunodeficiency virus, helicobacter pyrori; sex hormone Examples include hormones such as. Especially CRP. CRP is a serum protein that exhibits a precipitation reaction with C-polysaccharide of Streptococcus pneumoniae, and is usually present in a trace amount in serum. The increase in blood CRP concentration increases sharply when various inflammatory diseases occur. Therefore, CRP is measured as an inflammation marker for the diagnosis of diseases such as infectious diseases and inflammation.

Hereinafter, the invention will be described in detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples and the like.

(Example 1) Evaluation of non-specific reaction suppression of casein Na and evaluation of accuracy / simultaneous reproducibility In Example 1, evaluation of non-specific reaction suppression of casein Na and accuracy / simultaneous reproducibility of the measurement reagent of the present invention. Confirmed the evaluation.

1) Preparation of anti-CRP antibody sensitized cellulose particles Sensitization of anti-CRP antibody to cellulose particles was performed as follows. Cellulose particles with an average particle size of 330 nm (NanoAct®, sold by Asahi Kasei Fibers Co., Ltd.) are stirred in a buffer solution to a concentration of 0.1 w / v% and anti-CRP antibody at a concentration of 100 μg / ml, and then centrifuged. Anti-CRP antibody sensitized cellulose particles were washed and prepared.

2) Preparation of reagent for measurement Purified water, anti-CRP antibody, casein Na, saccharide, and surfactant were added to the anti-CRP antibody-sensitized cellulose particles obtained in 1) above to prepare a reagent for measurement. As a comparative example, a measurement reagent was prepared by adding BSA (bovine serum albumin) or skim milk instead of casein Na. The reagent composition of each measurement reagent is summarized in Table 1.

3) Preparation of measurement kit To the upper and lower parts of the reaction part of the measurement device, 0.45 μL and 0.5 μL of the measurement reagents prepared in 2) above were applied and air-dried at room temperature to prepare a measurement kit.

4) Preparation of CRP controlled sample
rCRP (sold by Oriental Yeast Co., Ltd.) was diluted with CRP-free serum (sold by Oriental Yeast Co., Ltd.) to obtain CRP controlled samples 0.4 mg / dL, 0.8 mg / dL, 1.6 mg / dL, and 3.0 mg / dL. The CRP-free serum and the prepared CRP-controlled sample were introduced into the sample supply section of the measurement device and mixed with the measurement reagent.

5) Measurement of absorbance
CRP-free serum and CRP-controlled sample are introduced into the measurement device, mixed with the measurement reagent, and then reacted at 37 ° C for 7 minutes. Photometry was performed at 810 nm and the absorbance after the reaction was measured. CRP-free serum and CRP-controlled sample were measured at N = 3, respectively.

6) Preparation of calibration curve A calibration curve was prepared from the average absorbance of the CRP-free serum obtained in 4) above and the CRP-controlled sample having a known CRP concentration (0.4, 0.8, 1.6, 3.0 mg / dL).

As a result, in Comparative Example 1 (BSA) and Comparative Example 2 (skimmed milk), a non-specific reaction was confirmed, and an ideal calibration curve could not be obtained. Anti-CRP antibody In Examples 1-1 to 1-4 in which the protein added to the sensitized cellulose particles was casein Na and the types of saccharides and surfactants were changed, no non-specific reaction occurred and CRP 0.0-3.0 mg / A concentration-dependent reaction was observed up to dL, and an ideal calibration curve could be drawn.

7) Evaluation of accuracy and simultaneous reproducibility Using control sample 1 (known concentration 0.41 mg / dl) and control sample 2 (known concentration 1.04 mg / dl) as test samples, 2 in the same procedure as 5) above. The concentration control sample was measured at N = 5, and the obtained absorbance was converted into a concentration from the calibration curve (Examples 1-1 to 1-4) prepared above, and the accuracy and simultaneous reproducibility were evaluated.

<Accuracy evaluation method>
For accuracy, the mean value measured at N = 5 and the known concentration were compared with the above two concentrations of CRP control sample, and the known concentration difference was calculated. It means that the accuracy is high when the known concentration is within ± 0.2 mg / dL.
<Evaluation method of simultaneous reproducibility>
For simultaneous reproducibility, the above two concentrations of CRP control sample were measured at N = 5, and the standard deviation (SD) was calculated. A standard deviation of 0.1 mg / dL or less means that the simultaneous reproducibility is high.

Table 2 shows the measurement results of the accuracy and simultaneous reproducibility of the control samples having the above two concentrations. In Comparative Example 1 (BSA) and Comparative Example 2 (skimmed milk), a non-specific reaction occurred, and the accuracy and simultaneous reproducibility could not be evaluated correctly. In Examples 1-1 to 1-4 to which casein Na was added, the known concentration difference was within ± 0.2 mg / dL and the standard deviation was 0.1 mg / dL or less for both of the two concentration control samples. Satisfied.

(Example 2) Confirmation of the optimum concentration of casein Na In Example 2, the optimum concentration of casein Na was confirmed for the measuring reagent of the present invention.

In Example 2, anti-CRP antibody-sensitized cellulose particles were specified as 0.2 w / v%, sucrose as 0.5 w / v%, CHAPS as 0.25 w / v%, and anti-CRP antibody as 100 μg / ml. ) Was 0.1w / v%, 0.2w / v%, 0.4w / v%, 0.6w / v%, 0.8w / v%, 1.00w / v%. ). The method for preparing the reagent for measurement was according to Example 1.

1) Preparation of calibration curve For the measurement reagents shown in Table 3, a calibration curve was prepared from the average absorbance of each test sample of CRP-free serum and CRP-controlled sample by the same procedure as in Example 1 above. A concentration-dependent reaction was observed at all concentrations of casein Na at CRP 0.0-3.0 mg / dL, and an ideal calibration curve could be obtained. In addition, the relationship between the mean value (measured value) converted from the above calibration curve and the CRP-controlled sample of CRP-free serum and CRP known concentration (0.4, 0.8, 1.6, 3.0 mg / dL) is shown in a graph (Fig.). 1). The average value (measured value) converted to concentration was close to the known concentration.

As described in detail above, according to the reagent for measuring turbidimetry and the measuring kit of the present invention, an ideal calibration curve can be drawn because non-specific reactions can be efficiently suppressed. It is possible to accurately measure the object to be measured in the test sample.

Claims (10)

A reagent for measuring an immunoturbidimetric method of a measurement target, which comprises carrier particles sensitized to a specific binding substance to the measurement target and casein or a salt thereof. The reagent for measuring turbidimetry according to claim 1, wherein the specific binding substance is an antibody or antigen that specifically binds to an object to be measured. The reagent for measuring an immunoturbidimetric method according to claim 1 or 2, wherein the carrier particles are any one of cellulose particles, latex particles, metal nanoparticles, and gelatin particles. 13. Reagent for measuring immunoturbidimetric method. The reagent for measuring turbidimetry according to any one of claims 1 to 4, further comprising a saccharide and / or a surfactant. The reagent for measuring turbidimetry according to any one of claims 1 to 5, wherein the measurement target is a C-reactive protein. The reagent for measuring turbidimetry according to any one of claims 1 to 6 is applied to a measuring device, and the measuring reagent is kept in a dry state until it is used for measurement. A kit for measuring the object to be measured by the immunoturbidimetry method. A reagent for measuring immunoturbidimetric method of the object to be measured, which contains carrier particles sensitized to a specific binding substance to the object to be measured and casein or a salt thereof, is applied to the device for measurement until it is used for measurement. A measurement kit by an immunoturbidimetric method of an object to be measured, characterized in that the measurement reagent is kept in a dry state. The measurement reagent applied to the measurement kit according to claim 7 or 8 is mixed with the test sample, and the carrier particles contained in the measurement reagent are subjected to a specific binding reaction of the measurement target in the test sample. A method for measuring an object to be measured by measuring the generated composite aggregate. A method for manufacturing a kit for measuring an object to be measured by the immunoturbidimetry method, which comprises the following steps:
1) A step of applying a reagent for measuring an immunoturbidimetric method of a measurement target, which contains carrier particles sensitized to a specific binding substance to the measurement target and casein or a salt thereof, to the measurement device;
2) A step of drying the measurement reagent applied to the measurement device.

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